random amplified polymorphic dna-based molecular ... the application of random amplified polymorphic

Download Random amplified polymorphic DNA-based molecular ... The application of random amplified polymorphic

Post on 29-May-2020




0 download

Embed Size (px)


  • Veterinary World, EISSN: 2231-0916 146

    Veterinary World, EISSN: 2231-0916 Available at www.veterinaryworld.org/Vol.12/January-2019/22.pdf


    Random amplified polymorphic DNA-based molecular heterogeneity analysis of Salmonella enterica isolates from foods of animal origin

    Surendra Singh Shekhawat1, Abhishek Gaurav1, Bincy Joseph2, Hitesh Kumar1 and Nirmal Kumar1

    1. Department of Veterinary Public Health and Epidemiology, College of Veterinary and Animal Science, Navania, Vallabhnagar, Udaipur, Rajasthan, India; 2. Department of Veterinary Microbiology, College of Veterinary and Animal

    Science, Navania, Vallabhnagar, Udaipur, Rajasthan, India. Corresponding author: Surendra Singh Shekhawat, e-mail: apsss14@gmail.com

    Co-authors: AG: gaurav.vets@gmail.com, BJ: dr.binzy@gmail.com, HK: hiteshkumar10121988@gmail.com, NK: dr.nirmalvet@gmail.com

    Received: 29-08-2018, Accepted: 03-12-2018, Published online: 26-01-2019

    doi: 10.14202/vetworld.2019.146-154 How to cite this article: Shekhawat SS, Gaurav A, Joseph B, Kumar H, Kumar N (2019) Random amplified polymorphic DNA-based molecular heterogeneity analysis of Salmonella enterica isolates from foods of animal origin, Veterinary World, 12(1): 146-154.

    Abstract Aim: This study aims to study the significance of random amplified polymorphic DNA (RAPD) typing in heterogeneity analysis of Salmonella serovars, isolated from foods of animal origin.

    Materials and Methods: Salmonella serovars isolated and identified from different foods of animal origin such as meat, milk, and egg by standard bacteriological methods. DNA isolated from all 10 isolates which are confirmed by biochemical and serotyping methods and then RAPD was performed using the primers OPB 10, primer 1290, NSC I, NSC II, and primer 3. Then, RAPD data were analyzed using the BioNumerics software, Belgium, Germany.

    Results: RAPD polymerase chain reaction (PCR) using five primers, namely OPB 10, primer 1290, NSC I, NSC II, and primer 3, classified the 10 isolates into 9, 10, 10, 7, and 10 RAPD-PCR types with discriminating powers of 0.1987, 0.423, 0.50889, 0.1842, and 0.2582, respectively. The phylogram constructed with NSC I profile classified isolates based on geographical origin. Primer 1290, NSC II, and primer 3 produced some uniform bands in all isolates indicating their binding ability in conserved genomic region. This study revealed that RAPD profile can be best used for finding out the heterogeneity at molecular level of Salmonella isolates in combination with other molecular and phenotypic typing techniques. Thus, our results support earlier observation of its significance by different workers on different Salmonella serotypes.

    Conclusion: Repeatability of RAPD-PCR is insufficient to distinguish genetic differences among Salmonella serovars.

    Keywords: Salmonella, random amplified polymorphic DNA, foods of animal origin, phylogram.


    Foodborne diseases are a serious a public health concern in food industry and Salmonella organisms are most frequently isolated bacterial agents of food- borne outbreaks [1]. Gram-negative Salmonella is a major health problem worldwide that causes typhoidal and non-typhoidal salmonellosis. Typhoidal and non-typhoidal illnesses cause millions of cases yearly with significant economic losses and even human deaths [2]. Most cases of non-typhoidal Salmonella (NTS) disease are associated with consumption of contaminated foods of the animal origin, particularly poultry meat and in some instances vegetables [3,4]. Salmonella serovars are responsible for infections occurring in developing as well as developed world and have been a major concern in terms of economic burden due to high morbidity [5]. Salmonella is an

    important cause of foodborne (alimentary) health problems in humans [6]. Salmonella was the second laboratory confirmed etiological agent accounting for 229 (30%) reported food poisoning outbreaks in the United States [7].

    The development of polymerase chain reaction (PCR) technology has allowed the specific amplifi- cation of particular target segments of DNA. Several PCR-based assays have been developed for rapid detection of Salmonella spp. [8]. Serotyping is most widely used phenotypic method, but it fails to provide appropriate information due to complex serotyping scheme and lack of comparison among different lab- oratories, thereby limiting its application to the refer- ence laboratories only [9]. Genotypic characterization of bacteria which are foodborne like Salmonella is very important to determine the genetic diversity of strains. Salmonella is a diverse group of bacteria with a large number of serotypes and strains present in var- ious hosts which include animal, birds, and humans. Moreover, due to diverse ecological habitats of this bacterium, there is a need of comparing the strains from different sources to determine the clonal varia- tion or similarity for physiological studies. Genotypic characterization helps to understand the complete

    Copyright: Shekhawat, et al. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

  • Veterinary World, EISSN: 2231-0916 147

    Available at www.veterinaryworld.org/Vol.12/January-2019/22.pdf

    epidemiology of the disease and aids in understand- ing the evolutionary pathways of various strains orig- inating from different ecological niches. Therefore, various novel genotyping methods such as variable number tandem repeats (VNTR), multilocus VNTR, multilocus sequence typing, and ERIC-PCR have been developed and utilized [10-13] to delineate epidemi- ological relationships among various isolates even within the same phage types [14]. However, the search continues for the easy to use efficient method capa- ble of differentiating strains of similar phenotype [15]. The application of random amplified polymorphic DNA (RAPD) analysis based on random amplification of genomic DNA fragments through short arbitrarily designed primers is an attractive alternative and has the potential to detect polymorphism throughout the entire genome as compared to other techniques [16].

    In the present study, we tried to analyze the sig- nificance of RAPD typing in heterogeneity analysis of Salmonella serovars, isolated from foods of animal origin. Materials and Methods Ethical approval

    In the present investigation, we have not used any live animals, therefore; no ethical approval was needed for the present study. Bacteria

    A total of 10 Salmonella isolates obtained from foods of animal origin [17] were used in the study. All the 10 positive Salmonella isolates were sent to Central Research Institute (CRI), Kasauli, Himachal Pradesh, for further serotyping. DNA isolation

    DNA isolated from all the 10 biochemically con- firmed isolates using HiMedia TM Bacterial Genomic DNA Purification Kit following the manufacturer’s instructions supplied along with the kit with suitable modifications. Briefly, 1.5 ml of overnight broth cul- ture was pelleted by centrifugation at 15,000 rpm for 2 min. The supernatant was discarded, and pellet was resuspended in 180 µl of lysis solution AL. After add- ing 200 µl lysis solutions C1, it was vortexed for 15 s and then incubated at 55°C for 10 min. Then, 200 µl of ethanol (95-100%) was added to the lysate and mixed thoroughly by vortexing for 15 s. The lysate so obtained was transferred into spin column and centri- fuged at 10,000 rpm for 1 min. The flow-through liq- uid was discarded and placed in a new 2 ml collection

    tube. Then, 500 µl of prewash solution was added to the spin column and centrifuged at 10,000 rpm for 1 min. The flow-through was again discarded, and same collection tube was used. A volume of 500 µl of diluted wash solution was added to column and centri- fuged at 15,000 rpm for 3 min and spin again at same speed for the additional 1 min to dry the column. The HiElute spin column was placed on a fresh tube and 100 µl of elution buffer which was kept in a water bath at 65°C for 30 min. The column was incubated at room temperature for 5 min followed by centrif- ugation at 10,000 rpm for 1 min. The spin column was then removed, and the collected DNA was stored at −20°C for further use. The concentration of DNA isolated was estimated spectrophotometrically using Biospectrometer (Eppendorf, USA) using the fol- lowing formula: DNA concentration (µg/µl) = [OD260 × dilution factor × 50 mg/ml]/1000. The purity was checked as ratio of OD260 and OD280, and the integ- rity of the purified DNA was assessed by running it in 0.7% agarose gel. RAPD-PCR

    RAPD-PCR was performed using genomic DNA of Salmonella isolates as a template with the random primers (Xcelris) mentioned in Table-1 [18-20]. Each PCR mixture consisted of 1 µg of template DNA, 2 pM solution of each primer (Xcelris, India), 5 µl 10× PCR buffer (Thermo Scientific, USA), 3 mM MgCl2 (Thermo Scientific, USA), 300 µM each nucleotides (Thermo Scientific, USA), and 3.75 units of Taq DNA polymerase (Thermo Scientific, USA) in 50 µl PCR reaction mix. PCR program for NSC I primer included initial denaturation at 94°C for 5 min, fol


View more >